Rheumatoid arthritis (RA) is a chronic progressive autoimmune disorder that ultimately leads to the destruction of the cartilage surrounding the joint. It is the second most common type of arthritis with symptoms first appearing in patients between 40 and 60 years of age. Current RA therapeutics can be classified into 3 groups: Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), corticosteroids, and Disease Modifying Anti-Rheumatic Drug's (DMARDs). The NSAIDs (e.g. aspirin, ibuprofen, naproxen) and corticosteroids encompass a large group of clinically effective compounds whose mode of action is well established—these compounds relieve pain and reduce inflammation by preventing prostaglandin synthesis through inhibition of cyclooxygenase 2 and the production of arachidonic acid, respectively. The DMARDs are an equally large group of therapeutics that includes both chemical (i.e., small molecules) and biological agents, e.g. antibody-based therapies. Examples of biological DMARDs include drugs such as etanercept, infliximab, and tocilizumab, which are therapeutically effective because they reduce the levels of inflammatory cytokines. Examples of chemical DMARDs include methotrexate, minocycline, and leflunomide. Interestingly, and in contrast to the well established modes of action of the NSAIDs, corticosteroids, and biological DMARDs, the molecular mechanisms by which the chemical DMARDs function as RA therapeutics are incompletely understood in several cases, e.g. minocycine.
Protein Arginine Deiminase 4 (PAD4), which catalyzes the conversion of peptidyl-arginine to peptidyl-citrulline, is widely believed to play a causative role in RA disease onset and progression because RA-associated mutations in the PAD4 gene have been identified in a variety of populations and RA patients produce autoantibodies that recognize citrulline-containing proteins. Interestingly, the anti-citrulline autoantibodies are considered to be the most specific diagnostic marker of this disease and there is a direct correlation between the levels of citrullinated proteins and disease severity, especially in the formative stages of RA. In total, the serological and genetic data suggest that PAD4 activity is dysregulated in RA, thereby suggesting this enzyme as a target for the development of a novel RA therapeutic.
While the development of the two most potent PAD4 inhibitors described to date have been reported, one or more of the aforementioned chemical DMARDs could inhibit this enzyme and thereby offer an explanation for their clinical efficacy. However, the standard PAD4 assay, which measures citrulline formation, is not readily amenable to high or even low throughput screens because it suffers from several limitations, including the fact that it requires the use of strong acids, toxic reagents, and high temperatures to convert the ureido group into a chromophore that absorbs light at 540 nm. Additionally, a number of compounds interfere with this assay, suggesting that potential inhibitors may be missed during the screening process. Therefore, a new inhibitor screen that remedies such shortcomings would be particularly beneficial.